Steroidogenic Factor 1 (SF-1) and CCAAT/Enhancer-Binding Protein b (C/EBPb) Cooperatively Regulate Progesterone Production

Presentation Number: MON-0380
Date of Presentation: June 23rd, 2014

Tetsuya Mizutani*1, Yoshitaka Imamichi1, Shinya Kawabe1, Shin Ishikane1, Tsukasa Osaki2, Naoto Minamino2 and Kaoru Miyamoto1
1Univ of Fukui, Japan, 2Natl Cerebral & Cardiovascular C, Osaka, Japan

Abstract

Steroidogenic factor 1 (SF-1, also known as Ad4BP) is a nuclear orphan receptor and plays a pivotal role in the regulation of reproductive and endocrine functions, including expression of steroidogenesis-related genes. Recently, it was found that SF-1 transduction in human mesenchymal stem cells (MSCs) induces cell differentiation into steroidogenic cell lineages. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by tandem mass spectrometry analysis was performed, and 24 proteins were identified. Among these proteins, we focused on CCAAT/enhancer-binding protein β (C/EBPβ), because C/EBPβ is known as a critical factor for ovulation, luteinization and steroidogenesis in the ovary, but little is known about the molecular mechanisms by which it regulates gene transcription in cooperation with SF-1.

To investigate whether C/EBPβ is involved in 8-Br-cAMP-induced steroidogenesis, progesterone production assays and gene expression analysis were performed. Knockdown of C/EBPβ dramatically attenuated the 8-Br-cAMP-induced progesterone production in granulosa tumor-derived KGN cells. Furthermore, the 8-Br-cAMP-induced STAR, CYP11A1 and HSD3B2 expressions were attenuated. EMSA and ChIP assays revealed C/EBPβ binding sites in the upstream regions of the STAR, CYP11A1 and HSD3B2 genes. These interactions were enhanced by cAMP stimulation. Interestingly, each C/EBPβ binding site is close to the SF-1 binding sites of these genes. Luciferase assay showed that C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated condition.

 

Nothing to Disclose: TM, YI, SK, SI, TO, NM, KM